U.S. patent application number 10/548588 was filed with the patent office on 2006-04-06 for dry polymer and lipid composition.
This patent application is currently assigned to CAMURUS AB. Invention is credited to Fredrik Joabsson, Helena Ljusberg-Wahren, Krister Thuresson.
Application Number | 20060073203 10/548588 |
Document ID | / |
Family ID | 9954828 |
Filed Date | 2006-04-06 |
United States Patent
Application |
20060073203 |
Kind Code |
A1 |
Ljusberg-Wahren; Helena ; et
al. |
April 6, 2006 |
Dry polymer and lipid composition
Abstract
The present invention provides an orally administrable
composition comprising a dry mixture of polymer, lipid and
bioactive agent, being capable on contact with water or GI tract
liquid of forming particles comprising said lipid and said
bioactive agent and optionally also water. It is preferable that
such particles have a liquid crystalline phase structure. The
invention also provides a method for the formation of compositions
comprising polymer, lipid and bioactive agent.
Inventors: |
Ljusberg-Wahren; Helena;
(Hollviken, SE) ; Joabsson; Fredrik; (Lund,
SE) ; Thuresson; Krister; (Bjarred, SE) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
CAMURUS AB
Ideon, Gamma 1, Solvegatan 41
Lund
SE
SE-233 70
|
Family ID: |
9954828 |
Appl. No.: |
10/548588 |
Filed: |
March 12, 2004 |
PCT Filed: |
March 12, 2004 |
PCT NO: |
PCT/GB04/01099 |
371 Date: |
September 12, 2005 |
Current U.S.
Class: |
424/469 ;
514/171; 514/54; 514/55; 514/57; 514/60 |
Current CPC
Class: |
A61K 9/19 20130101; A61P
15/08 20180101; A61K 9/2027 20130101; A61K 9/1075 20130101; C09K
19/3819 20130101; C09K 19/02 20130101 |
Class at
Publication: |
424/469 ;
514/057; 514/060; 514/171; 514/055; 514/054 |
International
Class: |
A61K 31/57 20060101
A61K031/57; A61K 31/716 20060101 A61K031/716; A61K 31/717 20060101
A61K031/717; A61K 31/728 20060101 A61K031/728; A61K 9/26 20060101
A61K009/26; A61K 31/715 20060101 A61K031/715 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 14, 2003 |
GB |
0305941.7 |
Claims
1. An orally administrable dry composition comprising at least one
physiologically tolerable polymer with dispersed therein particles
comprising at least one physiologically tolerable lipid and a
bioactive agent, which particles on contact with water or GI tract
liquid form cubic phase, hexagonal phase or L.sub.3 phase
nanometre-sized particles containing said lipid, said bioactive
agent and water and wherein said lipid comprises a diglyceride.
2. An orally administrable dry composition comprising a dry mixture
of at least one physiologically tolerable polymer, at least one
physiologically tolerable lipid and at least one bioactive agent,
said lipid, bioactive agent and polymer being interdispersed at a
molecular level and being capable on contact with water or GI tract
liquid of forming cubic phase, hexagonal phase or L.sub.3 phase
particles comprising said lipid and said bioactive agent and
optionally also water.
3. An orally administrable dry composition comprising at least one
physiologically tolerable polymer with dispersed therein particles
comprising at least one physiologically tolerable lipid and a
bioactive agent, which particles on contact with water or GI tract
liquid form nanometre-sized reversed hexagonal liquid crystalline
particles containing said lipid, said bioactive agent and
water.
4. A composition as claimed in claim 1 wherein said polymer is a
hydrophilic water soluble polymer.
5. A composition as claimed in claim 1 wherein said polymer is a
hydrophilic polymer capable of forming a gel when dissolved in
aqueous solvent.
6. A composition as claimed in claim 1 wherein said particles
contain water and are of a phase selected from normal cubic phase,
reversed cubic phase, normal hexagonal phase, reversed hexagonal
phase and L.sub.3 phase.
7. A composition as claimed in claim 1 wherein said particles have
a maximum dimension of 10 nm to 100 .quadrature.m.
8. A composition as claimed in claim 1 additionally comprising a
surfactant.
9. A composition as claimed in claim 8 wherein said surfactant is a
sugar surfactant.
10. A composition as claimed in claim 1 herein said particles are
surface modified with a surface active polymer.
11. A composition as calimed in claim 10 wherein said surface
active polymer is selected from chitosan, chitosan derivatives,
alginante, alginante derivatives, cellulose, cellulose derivatives
and mixtures thereof.
12. A composition as in claim 1 wherein said particles are
muco-adhesive.
13. A composition as claimed in claim 1 wherein said lipid
comprises a swelling polar lipid selected from gatactolipids,
lecithins, phosphatidylethanolamines, phosphatidylinositols,
phosphatidylserines, sphingomyelins, monoglycerides, acidic soaps,
cerebrosides, phosphatidic acids, plasmalogens, cardiolipins,
di-glycerolesters of fatty acids, oligo-glycerolesters of fatty
acids, poly-glycerolesters of fatty acids, di-glycerolethers of
fatty alcohols, oligo-glycerolethers of fatty alcohols
poly-glycerolethers of fatty alcohols and mixtures thereof.
14. A composition as claimed in claim 1 wherein said lipid
comprises at least one fatty acid.
15. A composition as claimed in claim 14 wherein said composition
releases particles in auqeous solution at pH below 3 and larger
particles in auqeous solution at pH above 6, wherein the particles
released at pH below 3 are sub-nanometer in size.
16. A composition as claimed in claim 1 wherein said polymer is
selected from polysaccharides, cellulose derivatives, cellulose
ethere, and synthetic polymers.
17. A composition as claimed in claim 16 wherein said polymer is a
polysaccharide selected from starch, maltodextrin, carrageenan,
xanthan gum, locus bean gum, acacia gum, chitosan, alginates,
hyaluronic acid and pectin.
18. A composition as claimed in claim 1 wherein said composition
forms 0.5 to 1000 nm particles upon exposure to aqueous fluids at
pH below 7 and 250 to 10 000 nm particles at pH above 6.
19. A process for the production of an orally administrable
composition, which process comprises removing solvent from a
solution of at least one physiologically tolerable polymer, at
least one physiologically tolerable lipid and at least one
bioactive agent, and optionally grinding, compacting, coating
and/or encapsulating the resultant solid.
20. A process for the production of an orally administrable
composition, which process comprises melting and mixing a mixture
of at least one physiologically tolerable hydrophilic polymer, at
least one physiologically tolerable lipid and at least one
bioactive agent, and optionally grinding, compacting, coating
and/or encapsulating the resultant solid.
21. A process as claimed in claim 20 wherein said melting and
mixing comprises melt extrusion.
22. A process for the production of a composition as claimed in
claim 1, which process comprises removing solvent from a solution
containing a dissolved physiologically tolerable water-soluble
hydrophilic polymer and a dispersed physiologically tolerable lipid
having dissolved or dispersed therein a bioactive agent.
23. A process for the production of an orally administrable
composition which process comprises removing solvent from a
solution containing a dissolved physiologically tolerable
water-soluble hydrophilic polymer, a dissolved or dispersed
bioactive agent and a dispersed physiologically tolerable lipid
wherein the lipid is dispersed in the solution in the form of
structured particles.
24. A process as claimed in claim 19 wherein the removal of said
solvent is carried out by lyophilisation or spray drying.
25. A pharmaceutical formulation comprising a composition as
claimed in claim 1 and optionally at least one pharmaceutically
acceptable carrier or excipient.
26. A pharmaceutical formulation as claimed in claim 25 comprising
a composition pressed into the form of a tablet.
27. A pharmaceutical formulation as claimed in claim 25 comprising
progesterone.
Description
[0001] This invention relates to orally administrable compositions
containing bioactive agents, e.g. pharmaceutical, veterinary, or
nutraceutical compositions, in particular compositions capable of
controlled release of the bioactive agent.
[0002] For many orally delivered compositions containing bioactive
agents, e.g. drugs, it is important that the agent be released from
the other components of the composition in a controlled or
sustained manner in order that the uptake of the agent from the
gastrointestinal (GI) tract should occur over a predetermined (e.g.
short or prolonged) period of time or in a particular region of the
GI tract.
[0003] The most widely practised controlled release technique
involves the use of compressed hydrophilic polymer matrices. Such
matrices form a gel layer on hydration within the GI tract. This
matrix can be erodible (e.g. soluble or biodegradable) or
non-erodible, and porous or non-porous, and the bioactive agent is
typically dissolved and/or dispersed in the matrix. Such
conventional controlled release techniques are described for
example in "Handbook of Pharmaceutical Controlled Release
Technology", Ed. Donald L. Wise, Marcell Dekker, New York,
2000.
[0004] Controlled release from non-erodible polymer matrices occurs
via dissolution of the bioactive agent followed by its
gradient-dependent diffusion through the gel layer, either through
the swollen polymer network itself or through solvent-filled pores
in the gel.
[0005] Where the bioactive agent is hydrophilic and highly soluble,
it can be difficult to achieve sustained release as the bioactive
agent is released relatively rapidly from the matrix. On the other
hand, where the bioactive agent is hydrophobic or poorly
water-soluble, it can be difficult to achieve a high degree of
release of the agent from the matrix and moreover there is a risk
that, once released, such agents may precipitate in the GI tract
with the result that uptake from the GI tract may be unpredictable
and highly variable.
[0006] In the alternative case of the erodible matrices, controlled
release of the bioactive agent is achieved through erosion of the
polymer matrix with the embedded bioactive agent being released
from the eroding surface. The release rate is thus mainly
determined by the rate of erosion of the matrix polymer. Highly
soluble, hydrophilic bioactive agents may also be released by
diffusion through the hydrated polymer matrix; however release by
diffusion is often negligible for poorly water-soluble or
hydrophobic bioactive agents. As with the non-erodible polymer
matrices, there is also the problem of precipitation of such
bioactive agents in the GI tract leading to unpredictable and
highly variable uptake of the agent from the GI tract.
[0007] We have now found that these problems of the conventional
controlled release techniques may be addressed by the use of hybrid
matrices comprising a polymer and a lipid which, on contact with
water, release self-assembled nanostructures, e.g. nanostructures
having a liquid crystalline structure.
[0008] Thus viewed from one aspect the invention provides an orally
administrable composition comprising a dry mixture of a
physiologically tolerable hydrophilic polymer (preferably a gelable
hydrophilic polymer), a physiologically tolerable lipid and a
bioactive agent, said lipid, bioactive agent and polymer being
interdispersed at a molecular level and being capable on contact
with water of forming particles comprising said lipid and said
bioactive agent and optionally also water.
[0009] The particles formed on contact with water are preferably
emulsion droplets, micelles, particles of inverse micellar phase,
vesicles, multilamellar bodies or aggregates or fragments of cubic,
L3, lamellar or hexagonal phase liquid crystalline structures. With
the lipid and polymer intermixed at the molecular level, such
particles will assemble automatically on contact with water or GI
tract liquids and will generally be nanometre-sized, e.g. with a
maximum dimension on the nanometre to micrometer scale, e.g. 0.5 nm
to 20 .mu.m, more typically 10 to 5000 nm, especially 100 to 1000
nm.
[0010] Interdispersion of lipid and polymer at the molecular level
cannot be achieved by techniques such as granulation, but
particularly effectively be achieved by solvent removal from a
solution of lipid and polymer in a common solvent or by mixing at
elevated temperature and/or pressure, e.g. by melt extrusion.
[0011] Whether or not interdispersion at the molecular level has
been achieved may readily be determined by scanning electronic
microscopy of the composition; where a large proportion, e.g.
>20% wt, of the lipid phase has collected as detectable
droplets, e.g. of 500 nm or larger (more preferably of 100 nm or
more), the admixing process will not have achieved the appropriate
molecular level intermixing. Following admixture of the lipid and
polymer at the molecular level, on storage some segregation may
occur. The dispersion of the components will still however be
superior to that achievable by granulation and the products are
deemed still to be in accordance with the invention.
[0012] In an alternative approach, the composition may take the
form of a polymer matrix containing pre-formed particles containing
bioactive agent and lipid which on contact with water form
(preferably liquid crystalline) nanoparticles, e.g. of L2,
L.alpha., L3 cubic, or hexagonal phase.
[0013] Thus viewed from a further aspect the invention provides an
orally administrable composition comprising a physiologically
tolerable water soluble, hydrophilic polymer with dispersed therein
particles comprising a physiologically tolerable lipid and a
bioactive agent, which particles on contact with water or GI tract
liquid form nanometre-sized particles (especially liquid
crystalline particles) containing said lipid, said bioactive agent
and water.
[0014] Again, by nanometre-sized is meant particles having a
maximum dimension on the nanometre to micrometer scale, e.g. 0.5 nm
to 20 .mu.m, more typically 10 to 5000 nm, especially 100 to 1000
nm. In an alternative aspect, nanometre-sized as used herein may
indicate particles on the nanomemter to micrometmer scale such as
10 nm to 100 .mu.m, more typically 50 nm to 10 .mu.m, especially
100 nm to 1 .mu.m.
[0015] In one preferred embodiment, the compositions of the
invention form small particles at low pH and larger particles at
higher pH. Specifically, upon exposure to aqueous fluids at pH
below 7, particularly below 3 and especially below 2.5, the
particles formed may be 0.5 to 1000 nm particles, preferably 10 to
500 nm, most preferably 10 to 200 nm. In contrast, upon exposure to
aqueous fluids at pH above 6.0, preferably above pH 7.0, particles
of size 200 to 100 000 nm, preferably 250 to 10 000 nm and most
preferably 400 to 5000 nm are formed (in some cases the particles
will be >1000 nm).
[0016] The production of compositions containing such pre-formed
liquid crystal precursor particles may be effected for example by
dispersing the lipid and the polymer in a liquid in which the
polymer is soluble but in which the lipid forms droplets, vesicles,
particles of liquid crystalline phase etc, and then removing the
solvent. The bioactive agent should be present dissolved or
dispersed in the lipid.
[0017] In the case of such compositions, contact with aqueous
fluids, e.g. the contents of the GI tract, causes the polymer
matrix to release lipid particles containing water and the
bioactive agent and having a liquid crystalline structure, for
example L2, L.alpha., cubic, L3 or hexagonal phase, i.e. they are
not simply structureless or water-unaffected droplets as in the
case of a (simple) oil-in-water emulsion.
[0018] The compositions of the invention may be produced using
appropriate combinations of components in order to achieve the
desired phase behaviour in the end product. How to select the
appropriate combinations is well within the normal capability of
the skilled person but nonetheless it may be helpful here to review
some simple rules in order to understand the phase behaviour of
lipids, surfactants, and other amphiphilic compounds. Rather than
specifying exact molecular structures or specific classes of
substances it should be understood that the teaching applies for
all compounds that are characterized by a bipolar structure with
hydrophilic and hydrophobic moieties localised at separated
positions. This provides this type of molecules with amphiphilic
properties such that the hydrophilic parts have a preference for a
polar environment while the hydrophobic parts have a preference for
a non-polar environment. This is the reason such molecules assemble
at interfaces between polar and non-polar regions and form
molecularly organised phases.
[0019] The phase behaviour of all amphiphilic molecules is governed
by the same type of physico-chemical rules. To be able to predict
the phase behaviour of a given surfactant or lipid or,
alternatively, to predict which compound to use to give the desired
phase behaviour, some empirical rules have been shown to be useful
(see Israelachvili, J. "Intermolecular and Surface Forces", 2nd
Edn., Academic Press, NY, 1991, and Jonsson et al. "Surfactants and
Polymers in Aqueous Solution", John Wiley & Sons, Chichester,
1998)
[0020] The "spectrum" of phase types can be considered to be
substantially as set out below.
CPP Value
[0021] TABLE-US-00001 Reversed micelles >1 Water-in-oil Cubic
Reversed hexagonal Cubic Lamellar 1 Mirror plane Cubic Hexagonal
1/3 to 1/2 Cubic Micelles <1/3 Oil-in-water
(where CPP, a dimensionless value, is v/1.a where v is the volume
of the hydrophobic component of the amphiphile, 1 is the extended
length of the hydrophobic component, and a is the maximum
cross-sectional area of the amphiphile. In this scheme, the
amphiphile can be considered to be conical in shape at either
extreme with the hydrophilic group at the cone base in the micelles
and at the cone point in the reversed micelles. On passing through
the "mirror plane" between the extremes, the lamellar phase, the
amphiphile can be considered to be cylindrical, i.e. its volume is
simply its length times its maximum cross-sectional area so
CPP=1).
[0022] The lamellar phase is often said to have a zero curvature,
since the amphiphile film has no preference to curve in any
direction. At the "oil-in-water" end of the scheme the structures
curve towards oil giving "normal" aggregates, while at the
"water-in-oil" end the structures curve towards water giving
"reversed" aggregates.
[0023] A strong tendency to form films with a high curvature gives
a preference for small spherical aggregates, like micelles, while a
less pronounced tendency for curved films may give larger and more
complicated aggregated structures. Thus, these are generally found
for amphiphilic compounds that have a preference to give films with
a curvature intermediate to that of micelles and the lamellar
phase.
[0024] One way to characterise an amphiphilic compound is by the
spontaneous curvature of the film. Its numerical value is
calculated as the inverse of the radius of the curvature of the
film. Essentially it can vary in between the inverse of the length
of the amphiphile molecule to a similar negative value (with the
lamellar phase at the mirror plane having a zero spontaneous
curvature). While the spontaneous curvature is a useful concept to
distinguish normal and reversed structures, it is not directly
related to the molecular structure of the polar lipid or the
amphiphilic compound in question.
[0025] A more useful way to be able to predict phase behaviour is
to use the "critical packing parameter" (CPP) concept. CPP is
calculated from geometrical considerations of the molecular
structure of the amphiphilic compound as mentioned above. v and 1
set limits on how fluid chains pack together, on average, in an
aggregate, and the mean molecular conformation thus depends on a,
v, and 1. It is important to recognize that by a is meant an
"effective" area. The relevance can be exemplified by the fact that
the head group repulsion between ionic surfactants is strongly
affected by screening electrolytes in a way such that it decreases
with increasing electrolyte concentration. This means that the same
ionic amphiphilic compound can, depending on the electrostatic
screening situation, give different structures. Analogous
situations are encountered with increasing temperature for
non-ionic surfactants having an oligooxyethylene containing head
group as well as for increasing concentration by themselves for
many surface active compounds.
[0026] As mentioned above, a normal spherical micelle has a
CPP-value below or equal to 1/3, the lamellar structure in the
mirror plane has a CPP.apprxeq.1, while the reversed structures are
characterised by CPP-values higher than unity. The more complicated
aggregated structures that typically are found in liquid
crystalline phases (e.g. cubic and hexagonal) have intermediate
CPP-values. For instance, a hexagonal structure has
1/3<CPP<1/2, while a bicontinuous cubic phase, which may have
a saddle-shaped geometry with two principal radii of curvature with
opposite sign, has a CPP-value close to unity.
[0027] As described above, surfactant geometry and packing
determine the aggregate structure, and often it is found that
single-chain surfactants form "normal" structures (e.g. micelles)
while double-chain surfactants or lipids have a preference to form
lamellar or reversed phases. It is also of utmost importance to
recognise that a desired phase behaviour (and effective CPP-value)
can be obtained by mixing two or more components of different
CPP-value.
[0028] In this context it also deserves mention that another
related empirical approach to characterise an amphiphilic molecule
is Bancroft's rule:--a water-soluble emulsifier tends to give o/w
emulsions, while an oil-soluble emulsifier tends to give w/o
emulsions. This rule of thumb is mainly used in emulsion technology
and has later been extended to the concept of
hydrophilic-lipophilic balance (HLB). Based on the molecular
structure, an amphiphilic compound can be assigned an HLB-number.
The HLB-number can be calculated by summation of the HLB group
numbers for the individual chemical groups that make up the
amphiphilic compound. The HLB number of an amphiphilic molecule can
then be used to predict whether normal or reversed emulsions are
likely to form.
[0029] Thus HLB group number for certain hydrophilic and lipophilic
groups are:
[0030] Hydrophilic Group Numbers TABLE-US-00002 --SO.sub.4Na 35.7
--CO.sub.2K 21.1 --CO.sub.2Na 19.1 --N (tertiary amine) 9.4 Ester
(sorbitan ring) 6.3 Ester (free) 2.4 --CO.sub.2H 2.1 --OH (free)
1.9 -0- 1.3 --OH (sorbitan ring) 0.5
[0031] Lipophilic Group Numbers TABLE-US-00003 --CF.sub.3 -0.870
--CF.sub.2.sup.- -0.870 --CH.sub.3 -0.475 --CH.sub.2.sup.- -0.475
--CH -0.475
and HLB is calculated as 7 plus the sum of the hydrophilic and
lipophilic group numbers. Where HLB is 3-6 the compound may find
use as a w/o emulsifier, 7-9 as a wetting agent, 8-18 as an o/w
emulsifier, 13-15 as a detergent, and 15-18 as a solubilizer.
[0032] For a system that gives "normal" structures, phase
transformations and/or disintegration of the aggregated structures
frequently take place on dilution with a large excess of an aqueous
phase. This is in contrast to the phase behaviour of amphiphilic
compounds that are characterised by reversed phases in equilibrium
with excess water. Such behaviour can be found for amphiphilic
molecules with CPP-values above unity. Both normal and reversed
phases can be used in the present invention.
[0033] Compositions according to the invention will typically be
produced as dry particulates: these can then be transformed into
desired solid dosage forms, e.g. by compressing into tablets
(optionally followed by coating, e.g. with a gastric acid resistant
coating), filling into capsules, granulation, pelletization,
grinding, etc. In such procedures, further components, e.g.
tableting aids, binders, flavours, aromas, sweeteners,
antioxidants, pH modifiers, viscosity modifiers, etc, may be added.
All such dosage forms qualify as compositions according to the
invention.
[0034] In the compositions according to the invention, which will
generally be pharmaceutical or veterinary compositions or
nutraceuticals, the bioactive agent may be hydrophilic,
hydrophobic, amphiphilic or a substance which is solubilized within
the GI tract, e.g. by virtue of the pH, intestinal flora, enzymes
or cell surfaces encountered therein. Within the compositions
themselves, the bioactive agent may be dissolved or dispersed
within the lipid and/or within the polymer or interdispersed with
either or both of these at the molecular level.
[0035] Preferably, the bioactive agent is dissolved or dispersed
within the lipid or interdispersed therewith at the molecular
level. This distribution of the bioactive agent may readily be
achieved by dissolving it in the lipid (where it is lipid soluble)
or by dispersing it within the lipid in particulate form, e.g. as a
water-in-oil emulsion where it is water-soluble or as a fine powder
(e.g. of nanoparticle size) where it is not lipid-soluble.
Alternatively it may be dissolved or dispersed in a solvent in
which the polymer and the lipid are soluble to form a solution or
dispersion of lipid, polymer and bioactive agent from which the
composition may be produced by solvent removal, e.g. by spray
drying, lyophilization or evaporation, for example under reduced
pressure.
[0036] Thus viewed from a further aspect the invention provides a
process for the production of an orally administrable composition,
preferably a composition according to the invention, which process
comprises removing solvent from a solution of a physiologically
tolerable hydrophilic gel-forming polymer, a physiologically
tolerable lipid and a bioactive agent, and optionally grinding,
compacting, coating and/or encapsulating the resultant solid. In
this method it is preferable that the solvent (which may, obviously
be a mixture of solvents) solubilises the polymer, lipid and
bioactive agent as a molecularly mixed solution. Advantageously,
the solvent should also be volatile to aid its removal from the
mixture. Examples of suitable solvents include water, ethanol,
isopropyl alcohol, formic acid, acetic acid (e.g. glacial or as a
mixture with water), dichloromethane, chloroform, acetone, ethyl
acetate and suitable mixtures thereof. Ethanol and acetic acid are
particularly suitable.
[0037] Viewed from another aspect the invention provides a process
for the production of an orally administrable composition,
preferably a composition according to the invention, which process
comprises melt extruding a mixture of a physiologically tolerable
(generally hydrophilic and gel-forming) polymer, a physiologically
tolerable lipid and a bioactive agent, and optionally grinding,
compacting, coating and/or encapsulating the resultant solid.
[0038] Viewed from a still further aspect the invention provides a
process for the production of an orally administrable composition,
preferably a composition according to the invention, which process
comprises removing solvent from a solution containing a dissolved
physiologically tolerable water-soluble hydrophilic polymer and a
dispersed physiologically tolerable lipid having dissolved or
dispersed therein a bioactive agent.
[0039] Viewed from a yet still further aspect the invention
provides a process for the production of an orally administrable
composition, preferably a composition according to the invention,
which process comprises removing solvent from a solution containing
a dissolved physiologically tolerable water-soluble hydrophilic
polymer, a dissolved or dispersed bioactive agent and a dispersed
physiologically tolerable lipid. The lipid is preferably dispersed
in the solution in the form of structured particles, especially
nanometer particles of liquid crystalline phase (as described
herein).
[0040] Besides lipid, polymer and bioactive agent, the solutions
used in the processes of the invention may desirably also contain a
surfactant with an HLB value in the range 8 to 18, e.g. a Tween,
Cremophor, Solutol, Brij, Triton, etc. The surfactant may be ionic
or nonionic, e.g. a sugar surfactant. Preferred sugar surfactants
include sugar (especially sucrose) fatty acid esters, especially
sucrose oleate, sucrose palmitate and/or sucrose laurate.
Particular mention may also be made of surfactants with large
polyoxyethylene head groups. Furthermore, to stabilise the (e.g.
liquid crystalline) particulate dispersions or emulsions, the
solutions used may desirably also contain an additional surface
active polymer, e.g. a starch or starch derivative, a copolymer
containing alkylene oxide residues (such as ethylene
oxide/propylene oxide block copolymers), cellulose derivatives
(e.g. hydroxypropylmethylcellulose, hydroxyethylcellulose,
ethylhydroxyethylcellulose, carboxymethylcellulose, etc) or graft
hydrophobically modified derivatives thereof, acacia gum,
hydrophobically modified polyacrylic acids or polyacrylates, etc.
The surface active polymer may also be used to provide a functional
effect on the surface of the particles, for example, in order to
selectively bind or target the particles to their desired site of
action. In particular, polymers such as polyacrylic acids or
chitosans may be used to provide mucus adhesive particles. Such
particles will thus tend to remain localised at their site of
release from the polymer matrix increasing the spacial control over
the active agent release. Compositions of the invention comprising
such surface modified particles form a further embodiment of the
invention.
[0041] One combination of lipid and surfactant of particular note
is the combination of a sugar surfactant (such as those indicated
herein supra) and lipids or lipid mixtures comprising mono-, di- or
tri-glycerides, particularly mono-glycerides and most preferably
glyceryl monooleate. These combinations show highly desirable
self-dispersing and self-emulsifying properties when used in the
compositions or methods of the present invention.
[0042] The polymer used in the preparation of the compositions of
the invention may be any polymeric material that serves to maintain
the composition in solid form before contact with water and which
serves to control the rate of release of lipid particles, e.g.
liquid crystalline nanoparticles, from the composition after
contact with water. A solid formulation (especially a free flowing
powder, which is a preferred form) is advantageous not only for
ease of dosing but also for ease of handling and processing during
manufacturing.
[0043] Examples of suitable polymers include water-soluble and
water-swellable polysaccharides (e.g. starch, starch derivatives
such as maltodextrin, carrageenan, xanthan gum, locus bean gum,
acacia gum, chitosan, alginates, hyaluronic acid, pectin, etc),
cellulose derivatives--in particular cellulose ethers (e.g. methyl
cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl methyl cellulose, hydroxyethyl ethyl cellulose,
ethylhydroxyethylcellulose, carboxymethyl cellulose, etc), and
synthetic polymers (e.g. polyacrylic acids, polyvinylpyrrolidone,
polyalkylene oxides (for example polyethylene oxides or
polyethylene glycols (PEGs)), etc.
[0044] The lipid used in the preparation of the compositions of the
invention may be any swelling or non-swelling polar lipid, e.g. as
defined by Small in the Journal of American Oil Chemists Society
45:108 (1968). Suitable examples of non-swelling polar lipids
include: diglycerides, triglycerides, fatty acids (e.g. C.sub.6-26
alkanoic and alkeneoic acids--the latter term including both singly
and multiply ethylenically unsaturated acids), waxes, sterol
esters, sterols (e.g. cholesterol, desmosterol, sitosterol, etc)
C.sub.6-26 alcohols, phytols, retinols, vitamins A, K, E and D,
etc.
[0045] Suitable examples of swelling polar lipids include:
galactolipids, lecithins, phosphatidylethanolamines,
phosphatidylinositol, phosphatidylserine, sphingomyelin,
monoglycerides, acidic soaps, cerebrosides, phosphatidic acid,
plasmalogens, cardiolipins, di-, oligo-, poly-glycerolesters of
fatty acids and -glycerolethers of fatty alcohols, etc.
[0046] The lipid, polymer and surfactant components of the
compositions of the invention may each be single compounds; however
it will generally be the case that two or more substances in one,
two or three of these three categories be used. Some substances may
fall into two or more such categories, e.g. polymers which are
amphiphilic or which contain an amphiphilic component, such as for
example acacia gum, may be used. These may play a dual or multiple
role in the compositions, e.g. providing desired solidity, release
profile, surface modification (e.g. for targeting or surface
adhesion) and lipid phase stabilization. In the case of the lipids,
it has surprisingly been found for example that the ability to load
the released lipid particles with certain hydrophobic and
amphiphilic bioactive agents is increased where the lipid comprises
as at least one of its components a saturated or a mono or
polyunsaturated C.sub.6-26 fatty acid or a salt, ester or ether
derivative thereof, e.g. oleic acid, linoleic acid, etc. or a salt,
ester or ether thereof.
[0047] On contact of the molecular level mixed lipid/polymer
compositions with water or gastrointestinal fluids, under the
action of the water the polymer may form a gel while the lipid
forms into particles containing lipid and bioactive agent. Where
the polymer (e.g. polymer gel) is erodible, the lipid particles
form and are released near the erosion boundary. Where the polymer
(e.g. polymer gel) is porous or porosified by one of the components
of the composition, lipid particles are formed within and diffuse
out through the pores, in some cases fully formed, in other cases
in forms which continue to take up water after release from the
polymer matrix.
[0048] The lipids in the molecularly mixed lipid/polymer
compositions may respond to water swelling of the polymer by
forming structured or non-structured lipid particles, e.g. L.sub.2,
L.sub..alpha., cubic or hexagonal phase liquid crystalline
nanostructures, or L.sub.3particles, micelles, microemulsion
droplets or amorphous structures. Formation of the non-structured
monoparticulates provides a particularly effective solubilizing
vehicle for hydrophobic and amphiphilic bioactive agents; formation
of L.sub.2, L.sub..alpha., cubic and hexagonal phase monostructures
(which have separate hydrophilic, hydrophobic and amphiphilic
microdomains) provides particularly effective solubilizing vehicles
for hydrophilic, hydrophobic and amphiphilic bioactive agents as
well as for mixtures of bioactive agents of different
hydrophilicities/hydrophobicities.
[0049] Where the lipid is entrapped within the polymer in the form
of pre-formed structured particles, these will consist of a liquid
crystalline phase, e.g. a fragmented inverse micellar (L.sub.2)
phase, a fragmented lamellar (L.sub..alpha.) phase, a fragmented
cubic phase, or a fragmented hexagonal phase. Such particles might
contain lipid, water and bioactive agent--in the case of the
L.sub.2, L.sub..alpha., L.sub.3, cubic or hexagonal phased
structures, the bioactive agent may be within a lipid or aqueous
domain within the structures or, for an amphiphilic agent, at the
boundary between such domains. However more generally the
structured particles entrapped within the polymer may take the form
of particles (e.g. solid, semisolid or fluid particles with a
crystalline or amorphous structure) which takes up water to produce
liquid crystalline nanostructures. The extent to which the
particles maintain a structured form will depend at least in part
upon the degree of drying carried out in order to render the
composition "dry" as considered herein. Where the particles lose
solvent to the extent that the original structure changes, exposure
to the water in biological fluids will cause the particles to
generate liquid crystalline nanostructures, for example with
L.sub.2, L.sub..alpha., L.sub.3, cubic or hexagonal phase
structure.
[0050] Thus contact of water with the lipid/polymer compositions
results in controlled (e.g. immediate, sustained and/or in use
regiospecific) release into the water (in use into the GI tract) of
lipid nanoparticles, generally 0.5 nm to 20 .mu.m, more typically
10 to 5000 nm, especially 100 to 1000 nm in mode maximum dimension,
containing the bioactive agent and from these, the bioactive agent
is released (e.g. into the water such as the GI tract contents) at
a rate that can be selected to optimize GI tract uptake and to
minimize precipitation or over-rapid release and uptake.
Alternatively, the rate of release can be controlled to give an
immediate release if desirable.
[0051] The compositions according to the invention may thus be
considered to comprise two essential sets of components: the
precursors of the lipid nanostructures that are released from the
composition on contact with water; and the precursors of the
release rate determining matrix. Besides this, the compositions may
of course, as mentioned above, comprise coating materials, binders,
flavours, preservatives, etc.
[0052] The lipid nanostructure precursor comprises one or more
bioactive agents, one or more lipids, optionally one or more
surfactants, and optionally water. The nature of the lipid
nanostructure released from the matrix is dependent on the physical
mode of incorporation of the lipid (i.e. admixed with the polymer
at the molecular level or embedded in the polymer as
nanoparticles), as well as the chemical composition of the
lipid/water/surfactant mixture. It is readily feasible to select
the chemical composition and manner of incorporation of the
precursor so as to cause the release of lipid nanoparticles of the
desired nature, e.g. by performing phase behaviour studies of the
reaction of the precursor components to water or other aqueous
media using standard techniques, e.g. as discussed in "The Aqueous
Phase Behaviour of Surfactants", R. G. Laughlin, Academic Press,
London, 1994.
[0053] The preparation of the lipid/polymer compositions of the
invention can be achieved by at least two preferred processes as
described above, i.e. by solvent removal from a solution of lipid
and polymer and where present surfactant (with the bioactive agent
dissolved or less preferably dispersed in the solution), or less
preferably by solvent removal from a dispersion of lipid in a
solution (generally but not essentially an aqueous solution) of the
polymer. In this latter case, the bioactive agent will be dissolved
or less preferably dispersed in the lipid phase which may also
contain surfactant and/or water.
[0054] In an alternative method, the solvent removal from a
dispersion of lipid in a solution (generally but not essentially an
aqueous solution) of the polymer may be carried out with the
bioactive agent dissolved in the polymer solution.
[0055] One advantage to the molecularly mixed state as opposed to
the entrapped nanoparticle state is that it is less sensitive, e.g.
to exposure to pressure during tableting or granulation.
Furthermore, the composition resulting from the solvent removal may
be the tablet precursor material, in which case this precursor
material is formed in a single step.
[0056] Solvent removal may be effected by conventional techniques,
e.g. solvent evaporation, lyophilization or spray drying, to give a
"dry" material which can if necessary be powdered, granulated,
tableted, coated, encapsulated, etc. to form solid dosage forms. By
dry it is meant that the material may be compressed to form
tablets. Preferably however a "dry" mixture, in powdered form, is
stable and free flowing.
[0057] Where components of the compositions of the invention are
heat sensitive, the solvent removal can be effected at ambient or
sub-ambient temperatures, e.g. by lyophilization. Where active
substances or excipients in the compositions are heat sensitive or
labile, solvent removal will generally be preferred over processes
involving elevated temperatures (e.g. melt extrusion) for the
preparation of the compositions of the invention.
[0058] Compositions according to the invention may also be prepared
by solvent removal (drying) from dispersions of lipid, polymer and
bioactive agent in rigorously degassed water. Such degassing
facilitates the mixing of the aqueous and non-aqueous phases and
may reduce the need for emulsifiers or stabilizers.
[0059] Where the lipid/polymer mixture is produced from a
dispersion of the lipid in a solution of the polymer, e.g. in water
or an organic solvent, the lipid may first be admixed with
bioactive agent and if desired surfactant and/or water before being
dispersed using conventional techniques, e.g. high speed mixing,
extrusion through a porous matrix, vortexing, sonication, high
pressure homogenization, microfluidization, rotor stator mixing,
etc. Dispersion will generally be into a solvent with the polymer
then being added and solvent removal subsequently being effected.
Where a surfactant is also added to the compositions, it is
possible for them to form self dispersing mixtures in which case
little if any mechanical dispersion will be necessary. Sugar
surfactants as described herein are suitable in this method. If
desired, e.g. to maintain a desired water content, the lipid may be
added in solid or semi-solid particulate form (produced for example
by cooling and pulverization or cold spraying), with the mixing
with a solvent and the polymer or with a polymer solution and the
subsequent solvent removal also being effected at sub-ambient
temperatures. The energy input during dispersion may be selected so
as to obtain the desired lipid particle size. If use of an organic
solvent is to be avoided, if no common solvent can be found, or if
incorporation of lipid nanoparticles with specific preferred
structures is desired, then this pre-dispersion technique is to be
preferred over the other technique involving solvent removal from a
solution of lipid and polymer. Once again, where any of the
components is heat sensitive, solvent removal is preferably
effected at ambient or sub-ambient temperature, e.g. by
lyophilization.
[0060] Where the bioactive ingredient has a tendency to
crystallize, e.g. on solvent removal or on cooling to sub-ambient
temperatures, the pre-dispersion technique may also be preferred as
the bioactive agent is more likely to become trapped in a
molecularly dissolved or solubilized form in the lipid
particles.
[0061] In a particularly preferred embodiment, using the
pre-dispersion technique, the bioactive agent may be dissolved in
the lipid at a level such that in the resultant lipid/polymer
mixture it is in a supersaturated state. Contact with fluids in the
GI tract then results in release of lipid nanoparticles containing
the bioactive agent in a metastable supersaturated state--such
nanoparticles have been found to exert a higher potency in
presenting the bioactive agent to the lining of the GI tract for
absorption as compared to lipid particles in which the bioactive
agent is in a normal stable state of dissolution. In a further
preferred embodiment, the bioactive agent may be dissolved in the
lipid at a level such that the resultant lipid/polymer mixture
contains bioactive agent as a thermodynamically stable solution but
generates lipid nanoparticles containing the bioactive agent in a
metastable supersaturated state upon contact with fluids in the GI
tract.
[0062] A further preferred technique for forming lipid/polymer
compositions of the invention is melting and mixing (e.g. by melt
extrusion or simply mixing at elevated temperature and optionally
elevated pressure). This method is advantageous in that it is quick
and simple to carry out, without requiring the removal of volumes
of solvent. It is most suitable for use when the bioactive agent is
not sensitive to elevated temperature, at least to the melting
point of the mixture. Obviously, a method comprising a mixture of
the "solvent removal" and "melt and mix" techniques may also be
used, in which the ingredients including a relatively small amount
of a suitable solvent (see supra) are mixed under somewhat elevated
temperature and preferably also elevated pressure. A suitable
mixture (e.g. a solution) may thus form with less solvent than
would be required at ambient or sub ambient temperature but the
temperature maintained lower than would be necessary for true
melting of the components. The solvent may then be removed by
reduction of pressure or by a later drying step using any suitable
technique (e.g. the techniques indicated supra for the solvent
removal method).
[0063] The compositions of the invention may also be formulated to
contain materials which porosify the polymer matrix or which serve
to produce gases on administration into the GI tract, e.g.
compounds which are more water soluble than the polymer,
fluorocarbons which are liquid below body temperature but gaseous
at body temperature, or gas generators such as sodium hydrogen
carbonate. These may facilitate lipid release or act to increase
the buoyancy of the composition causing prolonged retention of the
composition in the region of the GI tract where gas release occurs,
for example the stomach.
[0064] Alternatively, retention of the composition in the stomach
and/or control over the release profile may be achieved by trapping
gas directly in the dry powder as part of the manufacturing
process. This could occur, for example, as a result of including a
dispersion of immiscible low boiling solvent (e.g. a fluorocarbon)
in the mixture of solvent, polymer and active agent. When the bulk
solvent is removed, the volatile solvent may evaporate producing
pores, bubbles or other voids within the polymer. Similarly, gas
could be generated by chemical means and trapped within the polymer
matrix. After a porous matrix has formed, the initial gas may
optionally be replaced by others if desired. This might be used,
for example, to control the release profile of the active by
altering how readily the gas dissolves in the aqueous fluid of the
stomach. As the gas dissolves, the pores will more readily fill
with fluid.
[0065] Once a dry powder of the lipid/polymer-hybrid has been
obtained this can be further processed into solid dosage forms such
as tablets, pellets, granules or capsules by conventional
techniques, optionally using further excipients commonly employed
in solid dosage forms such as fillers, binders, disintegration
aids, glidants, lubricants, colours, flavours, sweeteners,
taste-masking agents, and film-coating materials. However, due to
the composition of the lipid/polymer-hybrid it is often not
necessary to add binders or lubricants since the polymer or,
respectively, lipid components of the lipid/polymer-hybrid are able
to act as such during e.g. tableting. Due to this reason
lipid/polymer-hybrid matrices are also particularly suitable for
direct compression.
[0066] The release profile for the bioactive agent over time can be
modified as desired by appropriate selection of the chemical nature
and molecular weight of the polymer (as is illustrated in the
Examples below). This is shown graphically in FIG. 1 of the
accompanying drawings which shows the different release profiles
for cyclosporin A (CsA) containing compositions in which the
polymer is respectively (o) low molecular weight PVP, (.cndot.)
high molecular weight PVP, (.diamond.) hydroxypropyl cellulose, and
(x) hydroxypropylmethyl cellulose. The cyclosporin A is released
from these compositions in the form of lipid carriers having CsA
therein. PVP is used herein to indicate polyvinyl pyrrolidone.
[0067] A further advantage of the lipid/polymer-hybrids is that
they are dry. By "dry", as used herein, is indicated that they are
functionally solid or semisolid, as opposed to fluid. Preferably,
dry, as used herein indicates that the compositions may be broken,
chopped, crushed or powdered, or otherwise formed into pieces of
controlled size and/or shape. Such pieces may then be processed to
enlarge, reduce or homogenise their granular sizes, coat them, mix
them with binders or other agents and render them suitable for easy
and handling in the manufacturing process (e.g as a uniform free
flowing powder). The term "dry" may, but need not, imply the
absence of solvents such as water and more generally indicates the
function of a material having the properties of a dry or solid
material. Thus, for example, a polymer matrix having trapped
therein liquid crystalline lipid/water/active agent particles may
function as a dry material in spite of the water content of the
liquid crystalline particles.
[0068] The dry nature of the compositions of the present invention
provides them with a considerable additional advantage. In
particular, compositions containing lipid excipients are generally
fluid, being, for example, in the form of an emulsion of active and
lipid in water or in the form of an oily lipid formulation, such as
an emulsion preconcentrate. Fluid formulations, however, present
additional problems in terms of packaging and distribution, dosage
and patient compliance when compared with dry formulations. Fluids
are more difficult for patients to carry, measure and take than dry
tablets and so a patient is less likely to comply correctly with
their treatment regimen if they are given a fluid rather than
tablets. Fluids may be packed into gelatin capsules but these are
complex to manufacture and often large and unpleasant to swallow.
The present dry formulations thus offer a considerable advantage in
ease of administration while preserving the other advantages of
lipid excipients. This advantage applies to "controlled" release
formulations of all types, whether the control is in the form of
immediate release (e.g. in the stomach) or whether the active agent
is released in a gradual, delayed or selective manner, in one or
more regions of the GI tract.
[0069] One challenge in formulation design is to find a composition
that is suitable for the active compound in question. Active
compounds can be sub-classed into three groups: hydrophilic
substances characterised by a high aqueous solubility; hydrophobic
(lipophilic) substances with low aqueous solubility but high
solubility in oils; amphiphilic or membrane soluble substances that
have a preference for interfaces between hydrophilic and
hydrophobic domains (including membranes).
[0070] In order to better understand the advantages and possible
uses of the present invention the three groups are briefly
discussed.
[0071] Solubility enhancers are generally not needed for the first
group and focus can be on obtaining a desired release profile. This
can be accomplished with standard techniques. However, some
hydrophilic substances with high aqueous solubility, e.g. peptides
and proteins, which are administrated via the oral route, are
sensitive to exposure to the hostile environment in the
gastrointestinal tract (pH, enzymatic activity etc.) or may suffer
chemical modification (e.g. ligand exchange). Others, e.g. heparin,
have difficulties in permeating the intestinal mucosal membrane.
The present invention can be used to overcome these problems. A
substance sensitive to gastrointestinal tract exposure can be
enclosed in hydrophilic domains within the lipid vehicles and in
this way be protected from degradation. Lipid vehicles released
from the solid matrices of the invention can also be designed to
include lipids that mediate uptake. Thus for instance capric acid
promotes absorption of large hydrophilic compounds, e.g. the
peptide desmopressin. Furthermore, the released lipid particles can
be surface modified by a suitable polymer (such as chitosan or
derivatives thereof) so as to provide muco adhesive or other
targeting properties. Another problem that can be encountered with
certain active substances is a variable aqueous solubility, i.e.
precipitating compounds (e.g. those which precipitate due to change
in pH or on contact with the calcium in the GI tract). The lipid
vehicles of the present invention can be used to either buffer a
local environment inside the lipid vehicles or to retain a local
milieu that promotes solubility of the active substances, also in
ambient media in which the active substance has low solubility.
Moreover, if transition to a state with low aqueous solubility
occurs the drug molecules can be solubilised in other domains of
the lipid vehicle.
[0072] The second group, comprising hydrophobic substances,
generally needs solubility enhancers to be presented to the
epithelial cells in sufficient quantities. This can be accomplished
by using lipid vehicles of the invention that contain hydrophobic
domains.
[0073] For the last class, amphiphilic or membrane soluble
compounds, the invention offers unique possibilities, since
nanostructure liquid crystalline phases are characterised by large
interfacial regions. Thus, amphiphilic substances can be
incorporated in high amounts resulting in high drug loads.
[0074] It should be noted that the advantages (protective
properties and mediated uptake) mentioned in connection with the
discussion about hydrophilic substances are also valid with the two
latter classes of active substances. It should furthermore be
recognised that substances sensitive to elevated temperatures,
which may degrade during manufacturing using standard processes,
e.g. melt extrusion, may conveniently be formulated and produced by
taking advantage of the present invention.
[0075] Examples of bioactive agents that can be used in the
compositions of the invention include but are not limited to
progesterone, cyclosporin A, cyclosporin G,
[O-(2-hydroxyethyl)-(D)Ser].sup.8-cyclosporin,
[3'-dehydroxy-3'-keto-MeBmt].sup.1-[Val].sup.2-cyclosporin,
bezafibrat, diltiazem, isradipin, verapamil, amphotericin B,
coenzyme QlO, danazole, atovaquone, amlodipine, nifedipine,
nimodipine, felodipine, paclitaxel, etoposide, irinotecan,
tretinoin, sirolimus, tacrolimus, itraconazole, ketoconazole,
propranolol, atenolol, atorvastatin, lovastatin, pravastatin,
simvastatin, enalapril, lisinopril, candesartan, losartan,
valsartan, olanzapine, sertraline, venlafaxine, mirtazepine,
raloxifene, sildenafil, tadalafil, clarithromycin, azithromycin,
ciprofloxacin, pioglitazone, rosiglitazone, atomoxetine,
cilostazol, celecoxib, rofecoxib, diclofenac, ibuprofen, naproxen,
aldosterone, betametasone, dexametasone, medroxyprogesterone,
prednisolone, diazepam, flurazepam, lorazepam, midazolam,
nitrazepam, and clomethiazol.
[0076] Especially conveniently the bioactive agent used in the
present invention is one having a solubility in water at 20.degree.
C. of less than 1% w/v, more especially less than 0.01% w/v.
[0077] We have also found that cyclosporins and cyclosporin
derivatives, in particular cyclosporin A, may particularly
advantageously be formulated for administration dissolved in a
fatty acid, e.g. a fatty acid containing up to 26 carbons, more
particularly 6 to 20 carbons, for example an unsaturated fatty
acid, especially a monounsaturated fatty acid, more especially a
C.sub.18 monounsaturated acid, more particularly oleic acid or also
favourably linoleic acid.
[0078] We have further found that cyclosporins and cyclosporin
derivatives, in particular cyclosporin A, may particularly
advantageously be formulated for administration dissolved in a
formulation comprising at least one fatty acid, e.g. a fatty acid
containing up to 26 carbons, more particularly 6 to 20 carbons, for
example an unsaturated fatty acid, especially a monounsaturated
fatty acid, more especially a C.sub.18 monounsaturated acid, more
particularly oleic acid or also favourably linoleic acid. The
formulations are preferably formulations according to the present
invention.
[0079] When compounds such as cyclosporins, cyclosporin
derivatives, cyclic peptides and in particular cyclosporin A are
fomulated as a composition of the present invention with a fatty
acid as indicated above then small (especially unimodal submicron)
particles have been observed to form upon contact with an aqueous
phase. These particles can contain a very high concentration of the
bioactive agent and are thus an advantageous method of delivery of
the bioactive since concentrated and supersaturated lipid
compositions are thought to provide enhanced uptake. The
formulation is also pH sensitive as indicated below.
[0080] In addition to giving an increased solubility to some
sparingly soluble bioactive agents (such as cyclosporins,
cyclosporin derivatives, cyclosporin A and certain cyclic peptides)
fatty acids also have the advantage that they can change property
depending on the surrounding pH. As a result, formulations
comprising fatty acids change properties such as their phase
behaviour, stability, solubility and such like depending upon the
pH of the region of the GI tract. A fatty acid containing dry
formulation, such as those described herein may thus, for example,
release small (especially submicron) particles at low pH (stomach),
while droplet size increases (e.g. to greater than half a micron,
especially to greater than 1 micron) at higher pH (like in
intestinal fluid). Destabilisation occurs at specific sites in GIT
and this destabilisation may be related to phase change of the
composition or released particles, precipitation and/or
supersaturation of bioactive agent. As a result, the inclusion of a
proportion of fatty acid in the lipid component of the formulations
of the invention may provide further control over bioactive agent
release and thus forms a further embodiment of the invention. The
compositions of the invention which vary in particle size release
with pH thus preferably contain a fatty acid.
[0081] In one embodiment of the invention, a composition of the
invention contains a fatty acid and releases particles in auqeous
solution at pH below 3 and larger particles in auqeous solution at
pH above 6, wherein the particles released at pH below 3 are
sub-nanometer in size. Preferably, the composition of the invention
contains components including sufficient fatty acid to provide
sub-micron (e.g. 0.5 to 1000 nm, preferably 1 to 250 nm, most
preferably 10 to 150 nm) particles upon exposure to pH below 7,
preferably below 3 and larger particles (e.g. 250 to 20 000 nm,
preferably 400 to 5 000 nm) at pH above 6.0, preferably above 7.
Such compositions may readily be prepared and identified by known
methods (such as laser light scattering) and by reference to the
Examples herein, especially Example 4 below.
[0082] We have also found that progesterone may particularly
advantageously be formulated for administration dissolved in a
C.sub.6-10 alkanoic acid, particularly caprylic acid or in
compositions comprising such fatty acids.
[0083] Thus viewed from a further aspect the invention provides a
pharmaceutical composition comprising progesterone or a derivative
thereof dissolved in a C.sub.6-10 alkanoic acid or a
physiologically tolerable salt thereof, said composition optionally
and preferably containing a further physiologically tolerable
lipid.
[0084] Such compositions may be formulated in any convenient dosage
form, e.g. capsules, solutions, powders, tablets, etc., and
conventional pharmaceutical carriers and excipients may be
used.
[0085] The compositions however are preferably formulated as orally
administrable compositions according to the earlier described
aspects of the invention.
[0086] The invention will now be described further with reference
to the following non-limiting Examples:
EXAMPLE 1
Release of Droplets without Internal Structure Containing
Progesterone from Formulations Solidified with Polyvinyl
Pyrrolidone (PVP)
[0087] Two compositions were prepared using low and high molecular
weight PVP (Plasdone K29/32 and K90 from ISP Technologies, Inc) and
were then lyophilized. The composition contents for the two
compositions are set out in Tables 1 and 2. TABLE-US-00004 TABLE 1
Composition before Composition after Substance lyophilization (%)
lyophilization (%) Progesterone 0.53 1.5 Glycerol dioleate (GDO)
2.1 5.9 Caprylic acid 2.1 5.9 Cremophor RH (CrRH) 4.2 11.8
Polyvinyl pyrrolidone (PVP) 26.7 75.0 K29/32 Ethanol (EtOH) 64.4
--
[0088] TABLE-US-00005 TABLE 2 Composition before Composition after
Substance lyophilization (%) lyophilization (%) Progesterone 0.19
1.5 Glycerol dioleate (GDO) 0.75 5.9 Caprylic acid 0.75 5.9
Cremophor RH (CrRH) 1.5 11.8 Polyvinyl pyrrolidone (PVP) 9.6 75.0
K90 Ethanol (EtOH) 87.2 --
[0089] Since ethanol solubilises the components in the formulation,
a molecular mixture was obtained. The formulation was solidified by
removing ethanol with lyophilization. This gave a solid formulation
which could be compressed to tablets (approximately 200 mg) by
compression in a KBr IR-tablet press.
[0090] The release profile was then studied using a simulated
intestinal fluid (SIF), pH=7.4, with the composition set out in
Table 3. TABLE-US-00006 TABLE 3 Substance Amount KH.sub.2PO.sub.4
34 g NaOH 7.8 g Deionized water 500 ml
[0091] The tablets were placed in baskets (rotating at 100 rpm) in
500 ml SIF at 37.degree. C. in an Erweka dissolution bath. Upon
contact with this excess aqueous phase, microemulsion droplets
without internal structure were released from the solid formulation
which carry the active substance. At each time where a data point
was obtained, aliquots were withdrawn (100 .mu.l) and analyzed with
HPLC to obtain the progesterone concentration in the dissolution
media at that time. The droplets had sizes below 1 .mu.m. The
release profiles seemed to be first order; and the release rate
could be controlled by changing the molecular weight of the
solidifying polymer. (Increasing the PVP molecular weight reduced
the release rate)
EXAMPLE 2
Release of Droplets without Internal Structure Containing
Cyclosporin A from Formulations Solidified with Polyvinyl
Pyrrolidone (PVP)
[0092] As in Example 1, two formulations were prepared, lyophilized
and compressed to tablet form using Plasdone K29/32 and K90. The
composition contents are set out in Tables 4 and 5 below
respectively. TABLE-US-00007 TABLE 4 Composition before Composition
after Substance lyophilization (%) lyophilization (%) Cyclosporin A
(CsA) 5.0 10.7 Maizine-35 5.8 12.5 Cremophor RH-40 7.1 15.3 Oleic
acid 2.1 4.4 Propylene glycol 1.6 3.5 Polyvinyl 24.8 53.6
pyrrolidone (PVP) (Plasdone K29/32) Ethanol (EtOH) 53.7 --
[0093] TABLE-US-00008 TABLE 5 Composition before Composition after
Substance lyophilization (%) lyophilization (%) CsA 1.8 10.7
Maizine-35 2.1 12.5 Cremophor RH-40 2.6 15.3 Oleic acid 0.75 4.4
Propylene glycol 0.59 3.5 Polyvinyl pyrrolidone 9.1 53.6 (PVP)
(Plasdone K90) Ethanol (EtOH) 83.0 --
[0094] Upon contact with an aqueous phase in excess using simulated
intestinal fluid (SIF) (as in Example 1) microemulsion droplets
without internal structure were released from the solid formulation
which carry the active substance. The droplets mainly had sizes
below 1 .mu.m. The release profiles were first order and the rate
could be controlled by changing the molecular weight of the
solidifying polymer. Again increasing molecular weight retarded
release.
EXAMPLE 3
Release of Droplets without Internal Structure Containing
Cyclosporin A from Formulations Solidified with Cellulose Based
Polymers
[0095] This Example shows that if a solvent cannot be found that
gives a molecular mixture of all the components that should be
included in the formulation, it may be feasible to produce a
polymer/lipid hybrid from an aqueous solution in which lipid
aggregates form. This route is expected to give a tablet in which
the lipid aggregates to some extent retain their structure from the
aqueous phase.
[0096] The composition of the formulation without the solidifying
polymer is given in Table 6. A high concentration of cyclosporin A
is enabled by using oleic acid in the formulation. 10% of the
formulation is emulgated in water and mixed with an aqueous
solution of a cellulose based polymer (1% (HPC) or 2% (HPMC)). The
mixture was lyophilized to obtain solid lipid formulations that
contained a 1:1 weight ratio polymer:formulation (see Table 7).
[0097] Tablets were prepared by compression in KBr IR-tablet press.
Upon contact with an aqueous phase (simulated intestinal fluid
(SIF)) in excess, drug containing droplets without internal
structure formed. The droplets mainly had sizes below 1 .mu.m. Drug
release profiles were obtained by using baskets (rotating at 100
rpm) in 500 ml SIF at 37.degree. C. in a Erweka dissolution bath.
At each time where a data point was obtained, aliquots were
withdrawn (100 .mu.l) and analyzed with HPLC to obtain the CsA
concentration in the dissolution media at that time.
[0098] As in the earlier Examples the release kinetics could be
controlled by changing the solidifying polymer. TABLE-US-00009
TABLE 6 Substance Amount (wt %) CsA 20.0 EtOH 13.3 Maizine-35 23.4
Cremophor RH-40 28.5 Oleic acid 8.3 Propylene glycol 6.5
[0099] TABLE-US-00010 TABLE 7 Substance Amount (wt %) CsA 11.5
Maizine-35 13.5 Cremophor RH-40 16.4 Oleic acid 4.8 Propylene
glycol 3.7 HPC or HMPC 50.0
EXAMPLE 4
Selection of Composition for Drying
[0100] The following example illustrates a procedure for selection
of drug composition that displays a phase change upon dilution in a
media where fatty acids dissociate but exhibits stability in acid
media where no such dissociation occurs. This formulation can be
dried for instance by the route outlined in Example 3.
[0101] A Cyclosporine containing liquid formulation (see Table 8a)
was manufactured in the following way. Cyclosporin A and ethanol
were weighed into a glass vial and closed with rubber stopper and
aluminium cap. The vial was placed on a rotating table until the
substance was dissolved into the ethanol. The rest of the
excipients were then added to the Cyclosorin A solution and the
vial was again closed and placed on the rotating table for at least
2 hours. The resulting liquid composition was inspected in
polarized light in order to determine that the sample was
homogenous and free of crystals before use. TABLE-US-00011 TABLE 8a
Substances Content % w/w Cyclosporin A 20 Ethanol 9 Propylenglycol
9 Oleic acid 19 Cremophore RH 40 44
[0102] The phase change of the samples was monitored as an increase
in particle size with time after dispersion in aqueous media. The
experiments were performed in the following way. Drops of the self
dispersing formulation were added directly on the surface of the
degassed aqueous medium in the sample compartment of the particle
sizer (Coulter LS230) and this dispersion procedure was continued
until the PIDS obscuration value exceeded 45%. The media used to
disperse the formulations were simulated gastric fluid (SGF) and
simulated intestine fluid (SIF). SGF was prepared by adding 2 g
sodium chloride and 7 mL hydrochloric acid into 1000 mL water, (pH
approximately 1.2) and SIF was prepared by adding 6.8 g potassium
phosphate and 190 mL 0.2M sodium hydroxide to 400 mL water,
followed by adjustment of pH to 7.5.+-.0.1 before the finally
dilute with water to 1000 mL. TABLE-US-00012 TABLE 8b Dispersion
Dispersion with pH 1.5 with pH 7.5 Particle size after 0 min. 126
nm 124 nm (mean) Particle size after 20 min. 124 nm 288 nm
(mean)
[0103] Measurements of particle size distribution were performed 0
minutes and 20 minutes after dispersion procedure was stopped. The
results of the experiments are summarized in table 8b above. This
table clearly shows that the mean particle size of formulation
dispersed in neutral pH increases with time but no change in mean
particle size is observed when the formulation is dispersed in acid
media.
EXAMPLE 5
Release of Particles of Fragmented Reversed Micellar (L.sub.2)
Phase Containing Progesterone or Cyclosporin A from Formulations
Solidified with PVP
[0104] The composition of the formulations are described in Tables
9a and 9b. TABLE-US-00013 TABLE 9a Substance Amount (wt %) CsA 5
Glycerolmonooleate (GMO) 20 GDO 25 PVP (Plasdone K29/32) 50
[0105] TABLE-US-00014 TABLE 9b Substance Amount (wt %) Progesterone
1.5 GMO 24.2 GDO 24.2 PVP (Plasdone K29/32) 50
[0106] On a weight basis, the formulations contain 50% wt of the
PVP polymer with low molecular weight (Plasdone K29/32). The dry
formulation was prepared in the following way: 1 wt % of the
formulation in Table 8, excluding the PVP, was dispersed in water
with the aid of sonication. To this solution the appropriate amount
of PVP polymer was added before thorough mixing and lyophilization.
After redissolution, lipid vehicles containing the drug substance
formed and had an internal structure. These were particles of a
fragmented reversed micellar (L.sub.2) phase. The size distribution
of the particles was not affected by the polymer or by the fact
that they were released from a tablet.
EXAMPLE 6
Release of Fragmented Lamellar Phase (Liposomes) Containing
Cyclosporin A from Formulations Solidified with Low and High
Molecular Weight PVP
[0107] This example illustrates that a molecularly mixed dry
formulation, that on contact with an aqueous phase releases a
fragmented lamellar phase (liposomes), can be obtained from mixing
the components in a common solvent. After mixing the components the
common solvent (EtOH) was removed by lyophilisation. The dry
formulation could be compressed to a tablet
[0108] The composition of the formulations before and after
lyophilization is given in Tables 10 and 11. TABLE-US-00015 TABLE
10 Composition before Composition after Substance lyophilization
(%) lyophilization (%) CsA 0.99 2.7 Diglycerol monocaprinate 8.02
22.3 (DGMC) Polyvinyl pyrrolidone 27.0 75.0 (PVP) (Plasdone K29/32)
Ethanol (EtOH) 64.0 --
[0109] TABLE-US-00016 TABLE 11 Composition before Composition after
Substance lyophilization (%) lyophilization (%) CsA 0.35 2.7
Diglycerol 2.86 22.3 monocaprinate (DGMC) Polyvinyl 9.65 75.0
pyrrolidone (PVP) (Plasdone K90) Ethanol (EtOH) 87.1 --
[0110] Upon contacting the dry formulation with an aqueous phase
(simulated intestinal fluid (SIF)) in excess, drug carrying
particles of a fragmented lamellar phase (liposomes) formed. The
size distribution of the liposomes was not affected by the polymer
or by the fact that they were released from a dry and compressed
tablet. Neither were the anisotropic properties of the liposomes
changed (as investigated with a microscope equipped with crossed
polarisers)
[0111] As in the earlier Examples, the release kinetics could be
controlled by changing the solidifying polymer. The higher the
molecular weight used, the slower the observed release.
EXAMPLE 7
Release of Fragments of a Lamellar Phase (Liposomes) from a
Formulation Solidified with PVP, and Control of the Size
Distribution of the Released Liposomes
[0112] Liposomes can be obtained with reduced particle sizes by
sonication before lyophilization. The reduced particle size is
conserved in the dry tablet form and reappears on redissolution.
Three size distributions were obtained in the following way. One
formulation was obtained by emulgating the composition given in
Table 12. TABLE-US-00017 TABLE 12 Substance Amount (wt %) CsA 10
Diglycerol monocaprinate (DGMC) 81 EtOH 9
[0113] A second by first emulgating the composition of Table 12 in
an aqueous solution that contained an appropriate amount of the PVP
polymer, followed by lyophilization. The resulting composition had
the constituents set out in Table 13. TABLE-US-00018 TABLE 13
Composition after Substance lyophilization (%) CsA 2.7 DGMC 22.3
Polyvinyl pyrrolidone 75.0 (PVP) (Plasdone K29/32)
[0114] A third composition, corresponding to the second in
contents, was subject to sonication before the lyophilization.
Sonication served to reduce the main mode released liposomal
particle diameter from about 4 .mu.m to about 0.4 .mu.m.
EXAMPLE 8
Effect of Decreased Size of the Liposomal Structures
[0115] In Table 14 is given the composition of a formulation that
on lyophilization gives fragmented lamellar (liposomal) structures.
The size distribution of the liposomes can be decreased by
sonication. Without sonification mode particle sizes were in the
range 0.1 to 2 .mu.m. With one minute sonication this was reduced
to a single mode at about 0.1 .mu.m. The formulation with the lower
size distribution gives a higher uptake in vitro in the Ussing
model as well as in vivo in the rat model using in situ perfusion
(illustrated in FIG. 2 of the accompanying drawings). In FIG. 2,
the time dependent .sup.3H--CsA absorption into the intestinal cell
membrane in the rat model using in situ perfusion of the proximal
small intestine is plotted as a function of dpm (disintegrations
per minute) against time for the composition of Table 14 without
sonification (o) and with one minute sonication (.cndot.)
TABLE-US-00019 TABLE 14 Substance Amount (wt %) CsA 10
Diglycerolmonooleate (DGMO) 42 Polysorbate 80 38 EtOH 10
EXAMPLE 9
Release of Bilayer Structures Containing Bioactive Agent
Amphotericin B from a Formulation Solidified with High Molecular
Weight PVP
[0116] An Amphotericin B containing formulation was prepared in the
following way;
[0117] (A) lyso-oleoyl phosphatidylcholine (LOPC, 24%),
phosphatidylcholine (PC, 38%), cholesterol (5.2%), and ethanol
(33%) were mixed over night to obtain a solution.
[0118] (B) High molecular weight polyvinyl pyrroloidone, Plasdone
K-90, ISP Technologies, Inc, PVP (10%) and water (90%) were mixed
over night to obtain a solution.
[0119] (C) Amphotericin B (AmB, 1.5%) was dissolved in a mixture
(90/10) of glacial acetic acid/water. After 30 minutes the mixture
was a homogeneous solution. The three solutions (A, B, and C) were
mixed in the proportions 13/74/13 and after thorough mixing to a
homogeneous solution the mixture was freeze dried to obtain a dry
formulation, Table 15.
[0120] 300 mg of the dry formulation was compressed to a tablet,
which was placed in a basket (rotating at 100 rpm) and released in
500 ml simulated intestinal fluid (SIF). The release of AmB in
fragmented bilayer carriers was monitored with UV-vis detection
operating at 415 nm and 500 nm. The released liposomal drug
carriers had a mean mode size below 1 .mu.m. TABLE-US-00020 TABLE
15 Composition before Composition after Substance lyophilization
(%) lyophilization (%) lyso-oleoyl 3.12 19.10 phosphatidylcholine
phosphatidylcholine 4.94 30.25 Cholesterol 0.68 4.14 EtOH 4.29 --
polyvinyl pyrroloidone 7.40 45.31 Plasdone K-90 Amphotericin B, AmB
0.195 1.19 glacial acetic acid 11.52 -- Water 67.88 --
EXAMPLE 10
Release of Cubic Phase Particles Containing Cyclosporin A,
Stabilised with Sucrose Ester of a Fatty Acid, from a Tablet
Solidified with Polyethylene Glycol.
[0121] This example illustrates that cubic phase particles can form
from a dry formulation in a self-dispersing process on contact with
an aqueous phase.
[0122] A molecular mixture was obtained by mixing the components in
Table 16. Two versions of the formulation were prepared differing
only by the fatty acid sucrose ester. The sucrose esters that were
used were either sucrose monopalmitate (Ryoto P-1570, Mitsubishi
Kagaku) or sucrose monooleate (Ryoto O-1570, Mitsubishi
Kagaku).
[0123] EtOH was evaporated from the molecular mixture to obtain a
dry powder that could be compressed to a tablet. After contacting
either of the two formulation with SIF (0.1%) a dispersion of cubic
phase particles was formed. The particles had a broad size
distribution (<100 .mu.m). TABLE-US-00021 TABLE 16 Composition
before Composition after Substance evaporation (%) evaporation (%)
cyclosporine A, CsA 2.5 10 fatty acid sucrose ester 2.5 10 (sucrose
monopalmitate or sucrose monooleate) glycerol monooleate, GMO 7.5
30 PEG (M.sub.w = 4000) 12.5 50 EtOH 75 --
EXAMPLE 11
Release of Cubic Phase Particles, Containing a Bioactive Agent
(Ketoconazole) with Weak Base Properties and Only Sparingly Soluble
in SIF, from a Formulation Solidified with PEG.
[0124] (A) 0.5 g ketoconazole (KC) was dissolved in 50 g glacial
acetic acid (HAc).
[0125] (B) 4.5 g of a solution consisting of 50% polyethylene
glycol (PEG, M.sub.w=35 000); 6% polysorbate 80 (P80); 6% Lutrol
F127; 2% oleic acid (OA); and 36% glycerol monooleate (GMO), was
molecularly mixed in the melted state at 70.degree. C.
[0126] The (A) and (B) solutions were mixed at 70.degree. C. to a
homogeneous solution. Glacial acetic acid was removed by
evaporation at 70.degree. C. By lowering the temperature a solid
formulation was formed, Table 17. The solid formulation was filled
in capsules; 0.650 g in each capsule.
[0127] Release profiles were obtained by contacting 2 capsules with
500 ml simulated intestinal fluid (SIF) at 37.degree. C. in an
Erweka dissolution bath (a basket rotating at 100 rpm was used).
Aliquots, 1 ml, were withdrawn at each time point and, after
molecularly dissolving the cubic phase particles having contained
therein KC by subsequently adding 0.75 ml 1.25M HCl in EtOH and
1.75 ml EtOH, the concentration of ketoconazole was assessed by
UV-VIS absorbance at 276 nm.
[0128] The release of cubic phase particles having ketoconazole
contained therein appeared 1st order and was finished after
approximately 1.5 hours. TABLE-US-00022 TABLE 17 Composition before
Composition after Substance evaporation (%) evaporation (%)
ketoconazole, KC 0.91 10.01 glacial acetic acid, HAc 90.91 -- PEG
(M.sub.w = 35 000) 4.09 44.99 polysorbate 80, P80 0.49 5.39 Lutrol
F127 0.49 5.39 oleic acid, OA 0.16 1.76 glycerol monooleate, GMO
2.95 32.45
EXAMPLE 12
Cubic Phase Particles Coated with Chitosan to Obtain a Positive
Surface Charge.
[0129] A melt mixture of 90% glycerol monooleate (GMO) and 10%
Lutrol F127 was dispersed in an aqueous solution with a homogeniser
to a course dispersion (5% lipid by weight). By using a high
pressure homogenisation (5 cycles at 5000 psi) the course
dispersion was transformed to a finer dispersion (mean mode size
200 nm after autoclavation for 20 min at 120.degree. C.). 20 ml of
an aqueous solution (5% acetic acid) containing 0.5% chitosan was
mixed with 20 ml of the 5% cubic phase lipid dispersion. This
dispersion was found to consist of cubic liquid crystalline phase
particles characterised by a mean mode size of 250 nm and a
.zeta.-potential of +4 mV in the pH range pH=4 to 6.5. Above pH 7
the particles became uncharged.
EXAMPLE 13
Release of Chitosan Coated Cubic Phase Particles from a Tablet
Solidified with PEG.
[0130] A melt mixture of 90% glycerol monooleate (GMO) and 10%
Lutrol F127 was dispersed in an aqueous solution with a homogeniser
to a course dispersion (5% lipid by weight). By using high pressure
homogenisation (5 cycles at 5000 psi) the course dispersion was
transformed to a finer dispersion (mean mode size 400 nm after
autoclavation for 20 min at 120.degree. C.). 20 ml of a solution
containing 0.5% chitosan (dissolved in an 5% acetic acid solution)
was mixed with 20 ml of the 5% dispersion of cubic phase particles
and 10 ml 10% PEG (M.sub.w=4000) solution. pH was adjusted to
pH=7.12 by titration with 1M NaOH. This dispersion was freeze dried
to obtain a dry powder which could be compressed to a tablet, Table
18. After dissolution in 500 ml simulated intestinal fluid (SIF),
cubic liquid crystalline phase particles with a broad size
distribution (<100 .mu.m) were obtained. TABLE-US-00023 TABLE 18
Composition before Composition after Substance lyophilization (%)
lyophilization (%) glycerol 1.8 42.86 monooleate, GMO Lutrol F127
0.2 4.76 Water 93.8 -- chitosan 0.2 4.76 HAc 2 -- PEG (M.sub.w =
4000) 2 47.62
EXAMPLE 14
Release of Chitosan Coated Cubic Phase Particles from a Tablet
Solidified with Polyethylene Glycol.
[0131] 4.5 g of a solution consisting of 50% polyethylene glycol
(M.sub.w=35 000), 6% polysorbate 80, 6% Lutrol F127, 2% oleic acid,
and 36% glycerol monooleate, was molecularly mixed in the melted
state at 70.degree. C. By lowering the temperature a solid
formulation was formed. The solid formulation was dispersed in
water (5% solid components) to form cubic phase particles. 20 ml of
a solution containing 0.5% chitosan (dissolved in an 5% acetic acid
solution) was mixed with 20 ml of the 5% dispersion of cubic phase
particles. pH was adjusted to pH=7.12 by titration with 1M NaOH.
This dispersion was freeze dried to obtain a dry powder, Table 19.
After dissolution in 500 ml simulated intestinal fluid, cubic
liquid crystalline phase particles with a broad size distribution
(<100 .mu.m) were obtained. TABLE-US-00024 TABLE 19 Composition
before Composition after Substance lyophilization (%)
lyophilization (%) Polysorbate 80 0.15 5.45 Glycerol 0.90 32.72
monooleate Lutrol F127 0.15 5.45 Water 94.75 -- chitosan 0.25 9.09
Glacial acetic 2.50 -- acid Polyethylene 1.25 45.45 glycol (M.sub.w
= 35 000) Oleic acid 0.05 1.82
* * * * *